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Novel Plague Virulence Factor Identified

By Duke Medicine News and Communications

DURHAM, N.C. -- Researchers at Duke University Medical Center
have identified a previously unknown family of virulence
factors that make the bacterium responsible for the plague
especially efficient at killing its host.

In the process, the team not only demonstrated that the use
of the common roundworm is a valid model for studying the
virulence of Yersinia pestis, the bacterium that causes plague.
They also showed that the interaction between Y. pestis and the
worm is quite similar to what occurs in mammals, including
humans. The work indicates that the pathogen may use similar
virulence mechanisms to infect evolutionary disperse
organisms.

These findings are important, the researchers continued,
since the pathogenesis system using the Caenorhabditis elegans
worm can accelerate the process of better understanding Y.
pestis pathogenesis. The shorter time and increased ease of
experimentation can be especially important, the researchers
continued, given potential use of Y. pestis as a biological
weapon, they said.

The results of the Duke research will appear Aug. 26, 2005,
in the on-line edition of European Molecular Biology
Organization (EMBO) Reports to be published in print in
October. The research was supported by the National Institutes
of Health's National Institute of General Medical Science, the
Southeast Regional Center for Emerging Infections and
Biodefense (SERCEB) and the Duke Center for Translational
Research.

"Our experiments have demonstrated how closely the Y.
pestis-C. elegans model we developed mimics what happens when
Y. pestis infects mammals," said Duke microbiologist Alejandro
Aballay, Ph.D., lead researcher of the team. "This system
should help speed the characterization of both pathogen and
host functions that potentially can be targeted for
intervention."

The Y. pestis bacterium primarily infects wild rodents, such
as mice, rats and squirrels. It is usually transmitted by
fleas, which spread the infection as they feed on the blood of
mammals. There are different forms of plague in humans --
bubonic, pneumonic and septicemic -- depending on site of
infection; and infections in humans are highly lethal if not
immediately treated.

"Dr. Aballay has developed a new model for dissecting the
ways pathogens such as the plague can infect and kill their
hosts," said Pamela Marino, Ph.D., scientist at the National
Institute of General Medical Science. "This creative approach
should improve our ability to develop new medicines to treat
such diseases."

Aballay has used the C. elegans, a worm commonly found in
the soil, as a model to study the virulence mechanisms of other
bacteria besides plague. The worm is an ideal model for genetic
studies, he said, because it takes only three days to develop
from an embryo to an adult capable of reproducing. Also,
scientists can easily manipulate specific genes in the worm,
and in contrast to other animal models, large quantities of the
worms can be grown quickly and can even be frozen and used
later.

"C elegans lives in the soil, so it continually comes into
contact with bacteria and other microbes," Aballay said. "It
has a highly developed system for not only recognizing
bacteria, but also responding to them. The ability of its
innate immune system to respond appropriately to specific
bacteria is very similar to that of mammals."

Aballay tested Y. pestis in his model because another
research team recently reported that the bacterium killed C.
elegans by creating a "biofilm" over the worm's pharynx,
causing it to die of starvation. Since mammals infected with Y.
pestis do not die in this manner, Aballay believed that other
virulence factors were involved in infecting the worm.

"We thought that a Y. pestis strain (known as KIM5) that
lacks the genes (hmsHFRS operon) required for biofilm formation
could still enter the worm's digestive system and eventually
kill it by using a method different from food blockage,"
Aballay explained. "We did in fact show that as Y. pestis
lacking the hmsHFRS operon accumulated in the intestine causing
a persistent and lethal infection."

The researchers then screened a library of almost 1,000 Y.
pestis mutants and found that six virulence factors are crucial
for the bacterium to have full virulence. Of the six virulence
factors, three are also required for infections in mammals. One
of these factors is similar to an exported protein of
Salmonella enterica. In a
study published last year, Abalallay showed that Salmonella
uses similar virulence factors to infect both mammals and C.
elegans.

"The protein produced by this new Y. pestis
virulence-related gene belongs to a family of uncharacterized
proteins found exclusively in pathogenic enterobacteria,"
Aballay said. "This work links for the first time this
particular family of bacterial proteins to virulence and we
have done so by using a C. elegans pathogenesis system and a
new mouse model of plague."

Aballay said that these types of conserved virulence factors
may regulate innate immunity in a broad variety of hosts,
including C. elegans, fleas and mammals.

"The importance of our work is that it will permit us to use
a model whose genetics can be easily manipulated as a viable
alternative not only for the identification of novel Y. pestis
virulence factors but also to study conserved innate immune
responses to the bacterium," Aballay said. "This may help us in
developing strategies to protect humans from the plague."

Since the KIM5 strain of Y. pestis is well characterized and
has a lower biosafety ranking than other infectious or toxic
agents, Aballay believes that it will much easier for research
laboratories to conduct research on virulence and innate
immunity.

Other members of the research team were Duke's Katie Styer,
Gregory Hopkins, and Richard Frothingham, as well as Sara
Schessar Bartra and Gregory Plano, University of Miami School
of Medicine.